Neutralizing epitope mapping of six beta1-bungarotoxin monoclonal antibodies and its application in beta1-bungarotoxin peptide vaccine design.
Twenty three stable monoclonal antibodies (mAbs) against beta1-bungarotoxin (beta1-bgt) were prepared by the hybridoma technique. Seven of the 23 mAbs (mAbs 2, 6, 8, 11, 17, 21 and 22) could inhibit more than 70% of phospholipase A2 activity of beta1-bgt and neutralize the toxin. Six of these neutralizing mAbs (mAbs 2, 6, 8, 17, 21 and 22) recognized continuous epitopes on the A chain of beta1-bgt and the other one (mAb 11) recognized a conformational epitope on the toxin. The continuous epitopes of these six mAbs were mapped using synthetic peptide and proteolytic enzymes. Experimental results indicate that mAb 17 recognized the A-chain residues 31-37; mAbs 2 and 8 recognized residues 46-51; mAbs 21 and 22 recognized residues 91-98; and mAb 6 recognized residue 100-106. The competitive-antibody-binding inhibition experiments showed that the affinity of these neutralizing mAbs to the native beta1-bgt is compatible with synthetic peptides. Furthermore, mice immunized with BSA-conjugated A-chain-peptide sequences A(31-37), A(46-51), A(91-98) or A(100-106) were protected from a high-dose beta1-bgt challenge. Subsequently, the peptide-immunized sera were passively injected into Balb/c mice and a significantly protective effect was also observed. To our knowledge, this study is the first systematic demonstration of multiple neutralizing B-cell epitopes of beta1-bgt, and this study is also the first report of the protective synthetic-peptide vaccine against beta1-bgt challenge.
Yang
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CC
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China.
Chan
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HL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000992:Antitoxins / Q000276:immunology*; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000276:immunology*; D018604:Epitope Mapping; D007116:Immunization, Passive; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010455:Peptides / Q000276:immunology
D000911:Antibodies, Monoclonal; D000992:Antitoxins; D002038:Bungarotoxins; D010446:Peptide Fragments; D010455:Peptides